This invention relates to nucleic acid probes for the detection of microorganisms. More particularly, the invention relates to a xe2x80x9creverse probexe2x80x9d nucleic acid probe.
Nucleic acid probes for the detection and analysis of microorganisms are known, although clinical applications of this technology are still comparatively rare.
These probes have clear potential value in the microbiology labs utilized in such disciplines as medicine, environmental and/or water resources engineering, agronomy and veterinary medicine. Certain probes and procedures have been developed for detecting the presence of microorganisms such as Chlamydia trachomatis, Haemophilus ducreyi, Mycobacterium and Escherichia coli, in clinical samples, environmental samples and the like.
A detailed review of gene probe technology is provided in the textbook xe2x80x9cGene Probes for Bacteriaxe2x80x9d (A. J. L. Macario and E. Conway de Macario, editors. Academic Press Inc. 1990. ISBN. 0-12-463000-6).
As a general overview, conventional gene probe techniques involve:
(a) selection of a nucleic acid probe (which may be based on the total nucleic acid of the species of interest; or some limited sequence thereof);
(b) affixing the nucleic acid from a sample onto a solid matrix (such as a nitrocellulose membrane);
(c) contacting the nucleic acid probe with the sample nucleic acid affixed on the matrix under conditions which permit hybridization; and
(d) detection of hybridization (as hybridization is regarded as evidence of the presence of the probe species within the sample).
Two of the most well known procedures used to probe microorganisms are:
(a) Southern blot (in which the sample nucleic acid is isolated, purified, subjected to restriction endonuclease digestion, subjected to electrophoresis, denatured, affixed to a solid matrix, then hybridized while affixed to the matrix with a radioactively-labeled probe); and
(b) dot or spot blot (in which lyzates of the sample, containing denatured nucleic acid, are affixed to the solid matrix without prior electrophoresis).
Thus, it will be apparent that the prior-art techniques require that the total nucleic acid from the sample be affixed to the matrix, in order to subsequently utilize a species specific probe. This procedure is very time consuming if it is desired to use a plurality of gene probes to attempt to characterize a sample (as a separate incubation is required for each gene probe employed).
The present invention mitigates certain of the disadvantages of the prior-art probe technology by reversing the sample/probe relationship.
Novel methods and compositions are provided for detecting the presence of a microorganism or a virus in a sample using nucleic acid isolated from the sample as a reverse genome probe. The method involves the steps of hybridizing the sample nucleic acid with a plurality of separately indentifiable nucleic acid standards prepared from organisms chosen based upon the origin of the sample, freeing the complexes of unbound sample and reagents, and detecting the complexes by means of a label attached to the sample nucleic acid. The indentity of the organism is obtained from the standard to which the sample nucleic acid hybridized. Optionally, the sample may be grown prior to analysis to increase the number of organisms. The method finds use in analyzing microorganism populations in environments where the microbial flora is poorly characterized.
Thus, in the broadest aspect of the present invention, a new and convenient method for the determination of the presence of more than one selected standard within a total microorganism sample is provided. In particular, the present invention provides:
In another embodiment of this invention, there is provided:
Claim to Master Solid Surface.
It will be appreciated that a xe2x80x9ctotalxe2x80x9d nucleic acid sample (i.e. a sample of the nucleic acid of the combined microorganisms present in the total sample) is employed as a reverse genome probe in the method of this invention.
As a further note of clarification regarding the difference between prior art gene probes and this invention, it will be recognized that the prior art probes may be prepared at and distributed from a central location (i.e. so that an end user provided with the probe then needs to prepare a filter or xe2x80x9csolid surfacexe2x80x9d in order to complete the prior art probing process) whereas this invention permits the preparation of a master solid surface at a central location (so that an end user provided with a master solid surface needs only to prepare the reverse probe in order to undertake the process of this invention).
In accordance with the subject invention, methods and compositions are provided for indentifying a organism in a sample, particularly in a field sample from an uncharacterized microbial environmental such as an oil field. The presence of an organism may be detected using nucleic acid prepared from the sample itself as a reverse gene probe.
This invention is generally suitable for gene probing a wide variety of prokaryotic microorganisms, including bacteria and eukaryotic microorganisms, including fungi, as well as viruses. As will be readily recognized by those skilled in the art, different types of gene probes have different sets of advantages and disadvantages. Although this invention provides a means to conveniently provide an initial characterization of a complex specimen or sample, this convenience may come at a cost of not being able to distinguish between microorganisms having very similar genomes (i.e. this invention does not allow one to completely distinguish between species having strongly cross-hybridizing genomes. However, where closely related organisms with homologous cross-hybridizing genomes may be present in the sample, and it is desired to obtain more specific information, the technique may be combined with other techniques known to those skilled in the art to identify more specifically any closely related organisms.) Accordingly, the term xe2x80x9cselected standardxe2x80x9d as used herein includes both:
(a) a microorganism having a genome that doesn""t cross-hybridize with any other; and
(b) a group of microorganisms having homologous, cross-hybridizing genomes (i.e. such a group represents a single selected standard).
In other words, a given selected standard may simply be one microorganism or it may be a group of closely related microorganisms. Thus, a given selected standard is different from another selected standard if they have genomes with little or no cross-hybridization.
In spite of this limitation, it will be readily apparent that the present invention provides a rapid means for the initial characterization of a sample, because only a single incubation (rather than multiple incubations) is required after a suitable master filter has been prepared.
This is because the xe2x80x9creverse probexe2x80x9d used in this invention is prepared using the total nucleic acid from the sample (whereas the probe of the conventional prior art technique is prepared with nucleic acid from the selected or target species). From this, it follows that the master solid surface used in this invention has affixed thereto spots of nucleic acid from selected standards of interest (whereas, as noted above, the prior art techniques involve the use of a solid surface having affixed thereto total nucleic acid from the sample and hence require an incubation with each conventional probe being utilized).
Additionally, it has been surprisingly discovered that the total specimen nucleic acid (i.e. nucleic acid obtained directly from the sample, without first isolating the species therein) is suitable for use as a xe2x80x9creverse genome probexe2x80x9d.
As noted above, the method of this invention generally involves three main steps:
1. Preparation of a Master Solid Surface.
2. Preparation of a Reverse Genome Probe.
3. Contacting the Reverse Genome Probe with the Master Solid Surface under conditions which permit hybridization.
Each of the above steps is described in detail below.
The master solid surface of this invention is similar to the solid surface of prior art probes, except for the fundamental difference that the solid surface of this invention is xe2x80x9cspottedxe2x80x9d with nucleic acid from the selected standards of interest, whereas the prior art solid surface is spotted with nucleic acid from the sample.
The material of the master solid surface is not critical to the success of this invention. Any of the materials which may be used to prepare conventional gene probe solid surfaces are suitable. Non-limiting examples of these materials include nitrocellulose, glass and nylon (including modified nylon filters).
The use of nylon is preferred, for convenience.
The initial step in preparation of the master solid surface is to obtain the selected standards of interest.
Individual cultures of each of these selected standards must then be made. As will be readily appreciated by those skilled in the art, this will typically involve making cultures of the bacteria or fungi of interest, or growing the virus of interest on a suitable host.
For each of these individual cultures, the following steps must be completed:
(a) isolating and purifying a representative nucleic acid sample, and
(b) affixing the representative nucleic acid sample to the master solid surface. (Note: as used herein, the term nucleic acid is meant to include deoxyribronucleic acid (DNA), ribonucleic acid (RNA), or both, as the context may require).
The isolation of the nucleic acid sample can be completed using procedures well known to those skilled in conventional gene probe techniques. It is preferred to utilize the genomic DNA (i.e. the complete DNA) as the representative nucleic acid of the selected standards in the preparation of the master solid surface.
The preferred procedure to isolate and purify nucleic acid involves:
(a) harvesting the cells from the culture (by centrifugation or filtration);
(b) lysing the culture, preferably with a cell wall degrading enzyme (such as lysozyme) and a detergent such as sodium dodecyl sulfate (or xe2x80x9cSDSxe2x80x9d) (optionally with the addition of Sarkosyl and ethylenediamine tetra-acetic acid (EDTA));
(c) treatment with enzymes (preferably proteinase K and/or ribonuclease A);
(d) solvent extraction (especially phenol and/or chloroform extraction); and
(e) precipitation (preferably with ethanol).
The resulting nucleic acid is subsequently dissolved in aqueous buffer in a defined concentration, denatured, spotted onto and affixed to a master solid surface.
As will be appreciated by those skilled in the art, the procedure used to affix the nucleic acid to the master solid surface will depend upon the type of solid material which is utilized.
For reasons of cost and efficiency, nylon is the preferred material for the master solid surface.
It will also be appreciated that the isolated, purified nucleic acid must be denatured (i.e. treated so as to separate the nucleic acid strands) prior to affixing it to the master filter.
The nucleic acid may be denatured, for example, by boiling or by treatment with sodium hydroxide (NaOH).
The resulting denatured nucleic acid is then affixed to the master solid surface, using conventional techniques. For a preferred nylon membrane master solid surface, this typically involves spotting a dilute solution of denatured nucleic acid from each of the species of interest at defined spots on the surface (i.e. so as to form a grid of the different selected species) followed by irradiation with ultraviolet light to link the denatured nucleic acid to the surface.
As previously noted, the reverse genome probes used in this invention are prepared using the total population of microorganisms present in the sample. Depending upon the application, it may be either necessary or desirable to increase the number of organisms present in the sample in order to have sufficient labeled nucleic acid to provide for a detectable signal. Accordingly, the sample may be grown in vitro using an appropriate nutrient medium to increase the microorganism population. Where the microorganism population in the sample has diverse nutrient requirements, aliquots of the sample may be grown in a plurality of nutrient media which contain different nutrients, for example carbon sources, and reverse nucleic acid probes prepared from each subculture. The total sample is subjected to the following steps to prepare the reverse genome probe:
(a) isolating and purifying the nucleic acid;
(b) labeling the nucleic acid.
Both of the isolation/purification and labeling steps can be completed using conventional procedures. The critical distinction over the prior art gene probe techniques is that the present invention requires the labeling of the entire nucleic acid of the sample, whereas the prior art technique labels only nucleic acid probes for the species of interest.
The preferred procedures for isolating and purifying the nucleic acid from the sample are essentially the same as the procedures used to isolate and purify the species-specific nucleic acid. The method of the present invention can be used for the detection of an organism in either the presence or absence of protein. However, when protein is present, an additional step to deproteinize the sample is desirable. Any conventional means can be used, for example phenol extraction, which does not adversely effect the integrity of the nucleic acid.
Generic methods of deproteinization may include mixing the sample with a suspension of glass particles in the presence of a high concentration of sodium iodide whereby DNA present in the sample is bound by the glass particles, isolating the glass particles, and then eluting the DNA with water or phosphate buffered saline. Glass particles may include finely ground glass beads (such as those sold under the name xe2x80x9cGeneClean IIxe2x80x9d by BIO/CAN Scientific Inc.).
Another method which can be used is admixing a protein-containing sample with a proteolytic enzyme composition, comprising, for example, at least one of the enzymes pronase or proteinase K. Following the enzymatic treatment, hydrolyzed product is removed, for example, by centrifugation.
After protein is removed, the nucleic acid is denatured, for example by heating at 90-100xc2x0 C., or treatment with sodium hydroxide or by other methods known to those skilled in the art. To prevent the nucleic acid from reannealing, the sample may be rapidly chilled or neutralized.
The isolated, purified nucleic acid from the sample is labeled to produce the reverse genome probe.
It is not intended to limit this invention to the use of any particular type of labeling procedure. To label the reverse nucleic acid probe, any of a variety of labels may be used including radioisotopes, fluorophors, or biotin. The label can be introduced to the nucleic acid by any standard enzymatic reaction, such as nick translation, or by terminal labeling, with 32P, 125I or biotin-labeled deoxynucleotide triphosphates (dNTP). The label generally is introduced prior to denaturation of the nucleic acid.
For laboratory use, the preferred procedure is to radiolabel the total nucleic acid from the specimen/sample with 32P by xe2x80x9cnick translationxe2x80x9d. Nick translation is a well known labeling technique which generally involves xe2x80x9cnickingxe2x80x9d the nucleic acid (with deoxyribonuclease-1) and xe2x80x9cextending the nicksxe2x80x9d (with DNA polymerase) in the presence of the radiolabeled nucleic acid precursors so as to incorporate the radiolabeled precursors in the newly synthesized nucleic acid.
Although radiolabeling is preferred for laboratory use, the use of a non-radioactive labeling technique is preferred for field use.
The (attempted) hybridization of the reverse sample genome probe and the master solid surface is undertaken using the hybridization techniques which are commonly utilized with conventional genome probes.
For the preferred nylon membrane master filter/radiolabeled reverse genome probe system, the hybridization technique simply involves contacting the master filter and the labeled, denatured reverse genome probe (preferably at an elevated temperature of between 40xc2x0 C. and 70xc2x0 C., for a period of 5 to 15 hours.
The probe is then removed from the filter, and the filter is carefully washed and air dried. X-ray film is contacted with and exposed to the probe, preferably for a period of 1 to 4 days. The presence of any dark spots on the developed X-ray film is indicative of hybridization between the reverse sample genome probe and the master filter (which, in turn, indicates that the species which was present on the master filter in the position(s) corresponding to the dark spot(s) on the X-ray film was present in the sample).
When it is desired to quantify the number of organisms present in a sample, at least one reference solution containing a known amount of nucleic acid representing a known number of organisms for hybridizing with a selected standard is treated identically to samples containing a known concentration of nucleic acid. At least one background solution containing no DNA is also included. The amount of label detectable in the background solution is subtracted from the amount of label detectable in the reference solution and the unknown sample. The relative amount of detectable label in the unknown sample thus indicates the relative amount of nucleic present in the sample and hence the number of organisms.
The reverse genome probe method finds use in analyzing microorganisms in environments with a microbial population that is not fully characterized. For example, a comparison of the organisms identified in corrosive and non-corrosive oil fields may aid in understanding the role of bacteria in the corrosion process. Also, a better understanding of the effectiveness of biocides on different bacterial populations in situ could be obtained when microorganism analysis is carried out sequentially on samples obtained from an oil field receiving biocide treatment. The technique may also be used to monitor the microbial diversity of an environment. Such information can be used to identify selected indicator organisms which are characteristic of a particular environment and whose presence can be monitored using more specific genome probes or other assays which will detect the specific organisms.
The method can also be generally applied toward the characterization or monitoring of microbial communities and other environments, including aquatic, soil, and animal. The technique may also find application in understanding and monitoring of large scale industrial processes that are catalyzed by microbial communities such as anaerobic sewage treatment. Suitable standards for a particular environment can be prepared from the organisms constituting the microbial flora in the environment under investigation. If the microbial population is analyzed without prior growth of sample, information concerning the population composition as it exists in nature may be obtained.